The present invention is directed to the field of diagnostic tests for detecting the presence of the fungus responsible for causing gummy stem blight in plants and seeds. More specifically, the present invention provides a method by which Didymella bryoniae, the causative agent of gummy stem blight, may be objectively detected and differentiated from related non-pathogenic organisms using a rapid assay based on the polymerase chain reaction.
Gummy stem blight, caused by infection with the fungal pathogen Didymella bryoniae, is a common disease affecting most cucurbits. Cucurbits refers to a botanical family, which includes among its members such agricultural crops as cucumbers, pumpkins, gourds, watermelons, cantaloupe, squash (summer and winter), and related plants. The fungus can infect both stems and leaves, in which case the disease is called gummy stem blight, or fruit, and is then referred to as black rot disease. The disease occurs worldwide and causes serious crop losses due to stem and fruit rot. In the United States, the pathogen is most common in the South, particularly the Southeastern regions where environmental conditions are ideal for the development and spread of the pathogen. In North Carolina, gummy stem blight is the second most important cucumber pathogen, and infection results in severe defoliation of plants in late production stages.
Traditional identification of fungal pathogens and subsequent diagnosis of plant diseases have usually involved growing the causative organism in pure culture, followed by characterization of the resultant colonies by examining spore morphology under a light microscope. However, such a diagnostic approach may be associated with technical problems. These include the inability to culture the disease-causing organisms, and the inaccuracy of diagnosis because closely related organisms may have similar morphological characteristics, making identification and differentiation between related species difficult. Further, the time required for culturing and differentiation of fungal pathogens ranges from 3 to 21 days. Thus, in addition to requiring a high degree of skill, such an approach to fungal identification may result in unacceptable time delays.
In the case of D. bryoniae, other problems exist which may make culture-based diagnosis difficult. For example, it has been reported that many D. bryoniae isolates fail to readily sporulate in culture and that those few that did sporulate seem to do so merely by chance. Therefore, traditional methods of identifying D. bryoniae may not always be successful, especially if sporulation is sporadic.
Additionally, several innocuous fungal species may be difficult to differentiate from D. bryoniae, thereby complicating diagnosis. For example, nonpathogenic Phoma species have also been isolated from tissues exhibiting gummy stem blight symptoms and are somewhat difficult to differentiate in the presence of D. bryoniae. 
As an alternative to diagnosis based on culture and spore morphologies, a technique based on random amplified polymorphic DNA analysis (RAPDs) has been developed to identify and differentiate D. bryoniae from related Phoma species. This RAPD technique uses random primers to generate small fragments of DNA of the organism in question, using the polymerase chain reaction. These fragments are then electrophoresed and the resulting pattern or xe2x80x9cfingerprintxe2x80x9d compared to other isolates, to aid in classifying the test organism and identifying unique areas of DNA (see A. P. Keinath et al, 1995, Morphological, Pathological and Genetic Differentiation of Didymella bryoniae and Phoma spp. Isolated from Cucurbits, 85 Phytopathology 364). However, this assay requires many different random primers to generate the fingerprint. Owing to the short primer lengths, the assay requires low annealing temperatures, which may cause variation in banding pattern and result in inaccurate diagnosis.
Therefore, there is currently an urgent need for an accurate diagnostic test for the identification of the pathogen D. bryoniae, especially for use in infected greenhouse transplants where the disease can spread very rapidly. There is also a need for an assay which can easily and rapidly differentiate non-pathogenic fungal species from the gummy stem blight pathogen. Further, there is a need for an assay to diagnose D. bryoniae from plant material (including leaves, stems, and seed), as well as differentiate D. bryoniae infection from non-pathogenic organisms which may be present.
The Polymerase Chain Reaction (PCR) is a technique by which a small fragment of deoxyribonucleic acid (DNA) can be rapidly duplicated, or cloned, to produce multiple DNA copies. The strength of the PCR technique is that it can be used to identify organisms from minute amounts of tissue samples because it proceeds in a series of cycles, with each successive round doubling the amount of DNA present in the sample. Thus, more than one billion copies of a single DNA fragment can be made in just a few hours, by mimicking the natural DNA replication process that occurs in living cells.
There are three phases essentially in a PCR reaction. In the first phase, denaturation, the original DNA extracted from the sample is heated to a temperature of from about 90xc2x0 C. to 95xc2x0 C. for a brief period, causing the individual DNA strands to separate. In the second or annealing phase, the temperature of the sample tube is lowered over a short period of time, allowing for the added oligonucleotide primers to bind to the separated DNA strands in a complementary fashion. In the final polymerization phase, the temperature of the sample mixture is again raised, to approximately 72xc2x0 C., allowing the polymerase enzyme to copy the DNA molecule rapidly. The three phases make up one complete PCR cycle, and take less than five minutes to complete.
The PCR reaction is repeated for a specified number of cycles, usually between 25 and 35, allowing the entire procedure to be completed in three to four hours. As an added advantage, this procedure can be automated with the use of commercially available thermal cyclers, allowing the entire procedure to be conducted using pre-determined parameters.
Following the completion of the PCR procedure, the samples may be run out on an electrophoresis gel to verify the presence of the desired DNA band. The electrophoresed products may be visualized using an ethidium bromide dye, or may be positively identified by hybridization with a probe specific for the bands of interest.
Over the past several years PCR technology has been shown to be applicable to the diagnosis of many human, animal, and plant organisms, and a variety of clinical assays have been evaluated. Results suggest that PCR is highly sensitive and, by varying conditions used, the technique can accurately discriminate between even closely related species.
PCR technology has never been used for diagnostic applications as in the present invention, because genetic sequences unique to D. bryoniae for use as primers have not been known. Nor have the primers and conditions suitable for differentiating closely related Phoma species from D. bryoniae been known.
By identifying specific primers unique to D. bryoniae and related Phoma sp., PCR technology can be used to provide an objective assay which overcomes some of the deficiencies of prior diagnostic methods for the identification and differentiation of D. bryoniae. Thus, the present invention allows for rapid diagnosis of gummy stem blight and requires only small amounts of fungal DNA infecting plant tissue or seed to provide a result.
The present invention recognizes and addresses the foregoing disadvantages and others of prior art constructions and methods.
Accordingly, it is an object of the present invention to provide a diagnostic assay for diagnosing gummy stem blight disease in plants.
Another object of the present invention is to provide a diagnostic test for gummy stem blight disease that is highly sensitive and rapid.
Yet another object of the present invention is to provide specific oligonucleotide primers which can be used to identify D. bryoniae DNA and differentiate it from closely related, non-pathogenic Phoma sp. DNA.
Still another object of the present invention is to provide a calorimetric assay for diagnosing gummy stem blight disease in plants.
It is still a further object of the present invention to provide a rapid diagnostic assay for determining the presence of Didymella bryoniae in plant tissues and seed.
It is yet another object of the present invention to provide a diagnostic test for differentiating the pathogenic fungus Didymella bryoniae from related, non-pathogenic Phoma species infecting plant tissues and seed.
These and other objects of the present invention are achieved by providing specific oligonucleotide primers in a PCR based assay to diagnose gummy stem blight in plants and differentiate the causative organism, D. bryoniae, from co-infecting nonpathogens.
The detection method utilizes the polymerase chain reaction, whereby DNA is extracted from potentially infected plant cells and is then replicated a number of times such that the original amount of DNA present is amplified greatly. The diagnostic assay requires the addition of unique primers, specific for D. bryoniae and for any organism desired to be differentiated from D. bryoniae, into a PCR reaction, thus resulting in enhanced amplification of a PCR product which can be putatively identified by visualization or definitively identified by hybridization with a specific probe.
For example, to identify the PCR product as D. bryoniae, a short oligonucleotide segment unique to D. bryoniae may be reacted with the PCR product and, if specific binding occurs, the PCR product is identified as D. bryoniae. Further, by reacting the product with probes for other organisms, the infective agent may be differentiated from co-infecting species, based on a negative hybridization result.
The PCR process is initiated by isolating genomic DNA from potentially-infected plants to serve as a template during the PCR amplification. Specific oligonucleotide primers are allowed to hybridize, or bind, to areas which are complementary in the target DNA. The primers then serve as sites to allow the polymerase enzyme to fill in the area between the bound primers, resulting in a doubling of the DNA. By repeating this cycle many times, a billion or more copies of the original DNA can be made. Specificity of the amplification procedure is controlled by the uniqueness of the primers, and the conditions of the PCR reaction.
Following PCR amplification, PCR products are electrophoresed into agarose gels, along with a DNA ladder for determining the size of the PCR product. The PCR product can be putatively identified using visual comparison to a size marker, or can be definitively identified by means of hybridization with a specific probe.
The present diagnostic method will allow for early detection and differentiation of fungal lesions on the leaves and stems of plants, or infection of seeds. Because PCR technology can amplify very small quantities of fungal DNA, the presence of D. bryoniae or other organisms can be detected in amounts so small that no visible sign of infection is present. Such early detection allows for rapid implementation of control measures, such as fungicide spraying in production fields, and permits identification of infected seed before marketing.
As discussed above, the present invention consists of a method for detecting D. bryoniae, and for differentiating this pathogenic organism from closely related non-pathogenic Phoma species, by detecting a unique segment of fungal DNA in plant tissues and seeds. The present method is more rapid, and may be more sensitive, than identifying D. bryoniae in pure culture and examining spore morphology under the light microscope. Further, the present method is less time consuming and labor intensive than using RAPD fingerprint analysis to differentiate between D. bryoniae and other Phoma species, since many different random primers have to be used to verify the accuracy of the results obtained with the RAPD technology.
The present method is also more cost effective when compared with currently used methods. Culture methods may require large amounts of special media and a high degree of skill to differentiate morphological characteristics of cultured organisms. Further, co-infecting organisms may not grow in the media chosen, resulting in inaccurate results. In contrast, the present diagnostic assay permits the simultaneous amplification of multiple samples and can be accomplished using very small amounts of fungal DNA, on the order of 50 nanograms (ng) or less.
Broadly speaking, the diagnostic method involves extracting genomic DNA from cells found in plant tissue, such as stems and leaves, or seed; replicating the DNA template with the use of specific oligonucleotide primers; amplifying the complimentary DNA for approximately 30 cycles of thermal cycling; and producing the PCR products which can be identified based on size or on hybridization ability with an oligonucleotide probe specific for D. bryoniae or other fungal species.
In summary, the present method of detecting gummy stem blight, using unique oligonucleotide primers to amplify a D. bryoniae sequence and differentiating D. bryoniae from closely related, non-pathogenic Phoma species, could greatly increase early treatment and control of gummy stem blight in greenhouses, commercial production fields, and seed production fields.